Vaccines are commonly formulated using an adjuvant. A common adjuvant-type is the aluminum based colloids, usually referred to as alum. More specifically these are usually aluminum hydroxide (aluminum oxyhydroxide) (also called alhydrogel) and aluminum phosphate (also called adju-phos). For example, vaccines useful against organisms such as anthrax (Bacillus anthracis) are commonly formulated with alhydrogel, which binds the anthrax antigen used in such vaccines (so called subunit vaccines). One aim of such product formulations was to maximize the binding of rPA to the alum. Since phosphate ions were known to desorb antigen from the alhydrogel colloid, the phosphate concentration in such formulations has been kept deliberately low (at 0.25 mM). At this concentration the phosphate buffer does not interfere with rPA binding to the alhydrogel colloid. For example, with this formulation we have found that recombinant protective antigen (rPA) binding was >98%.
Batches of this drug product formulation have been used in the Phase I and Phase II clinical studies, which demonstrated safety and immunogenicity in man.
With this original formulation the pH was found to be 5.9 due to the insufficient buffering capacity of the low phosphate buffer. In order to increase the stability of the rPA drug product and provide a more physiological pH, it was decided that increased control of the formulation was required. The pH of the drug product formulation had to be increased to pH 7 so that an improvement in the buffering capacity of the formulation would be required. For control at pH 7.0, a possible physiological buffer is phosphate, which has a pKa at 7.2 but the disadvantage of using phosphate in the formulation was the inhibitory effect upon rPA-alum binding.
Based on pKa, the possible alternative would be histidine (pKa 6.04), however when stored as a liquid, it has the propensity to oxidize and produce a brown coloration. Moreover, introduction of an alternative buffer such as histidine would mean a radical change in the formulation. A new approach was therefore needed.
We subsequently discovered that we could increase phosphate concentration to a level that both controlled pH and afforded minimal effect on rPA:alhydrogel binding. Consequently, the phosphate concentration was increased to approximately 4 mM; a concentration that was capable of maintaining the Drug Product at a pH of approximately 7.0, and did not markedly affect the amount of unbound rPA, which remained below the level of detection of the assay (<2%).
This led to the surprising result that such a phosphate concentration also greatly increases the bioactivity of the vaccine formulation.